Subcellular localization analysis of the closely related Fps/Fes and Fer protein-tyrosine kinases suggests a distinct role for Fps/Fes in vesicular trafficking

The subcellular localizations of the Fps/Fes and closely related Fer cytoplasmic tyrosine kinases were studied using green fluorescent protein (GFP) fusions and confocal fluorescence microscopy. In contrast to previous reports, neither kinase localized to the nucleus. Fer was diffusely cytoplasmic throughout the cell cycle. Fps/Fes also displayed a diffuse cytoplasmic localization, but in addition it showed distinct accumulations in cytoplasmic vesicles as well as in a perinuclear region consistent with the Golgi. This localization was very similar to that of TGN38, a known marker of the trans Golgi. The localization of Fps/Fes and TGN38 were both perturbed by brefeldin A, a fungal metabolite that disrupts the Golgi apparatus. Fps/Fes was also found to colocalize to various extents with several Rab proteins, which are members of the monomeric G-protein superfamily involved in vesicular transport between specific subcellular compartments. Using Rabs that are involved in endocytosis (Rab5B and Rab7) or exocytosis (Rab1A and Rab3A), we showed that Fps/Fes is localized in both pathways. These results suggest that Fps/Fes may play a general role in the regulation of vesicular trafficking.     

Involvement of Fes/Fps tyrosine kinase in semaphorin3A signaling

Collapsin response mediator proteins (CRMPs)/TOAD64/Ulips/DRPs and CRAM have emerged as strong candidates for a role in semaphorin signaling. In this study we identified Fes/Fps (Fes) tyrosine kinase in the CRMP-CRAM complex and investigated whether Fes was involved in semaphorin3A (Sema3A) signaling. In COS-7 cells, the interaction between Fes and plexinA1 (PlexA1) and the tyrosine phosphorylation of PlexA1 by Fes were observed; however, these events were significantly attenuated by co-expression of neuropilin-1 (NP-1). Even with NP-1 co-expression, Sema3A was able to enhance the association of Fes with PlexA1 and Fes-mediated tyrosine phosphorylation of PlexA1, CRAM and CRMP2. Co-expression of Fes with PlexA1 exhibited COS-7 cell contraction activity, indicating that Fes can convert inactive PlexA1 to its active form, whereas combination of Fes/NP-1/PlexA1 or Fes kinase-negative mutants/PlexA1 did not alter cell morphology. Finally, Sema3A-induced growth cone collapse of dorsal root ganglion neurons was suppressed by expression of Fes kinase-negative mutants. Taken together, our findings suggest that Fes links Sema3A signals to CRMP-CRAM, and that NP-1 negatively regulates PlexA1 activation by Fes in resting condition.     

Disruption of coiled-coil domains in Fer protein-tyrosine kinase abolishes trimerization but not kinase activation.

The protein-tyrosine kinase Fer and the highly homologous proto-oncoprotein Fps/Fes are implicated in signaling from a variety of growth factor and cytokine receptors. Here we examine the molecular basis of Fer kinase activation with an emphasis on the role of oligomerization. We show that Fer forms trimers in vivo and that disruption of either the first or second coiled-coil domain abolishes oligomerization, suggesting a cooperative interaction between these two domains. Although Fps/Fes also forms homotypic oligomers, probably via homologous coiled-coil domains, no heterotypic interactions were observed between Fer and Fps/Fes. Incorporation of catalytically inactive Fer peptides into the oligomeric complex caused only mild reduction of wild type Fer kinase activity, suggesting that kinase-inactive Fer would not behave as a potent dominant negative. Although oligomerization of Fer can potentiate autophosphorylation in trans at three major phosphorylation sites, these residues can likely also be phosphorylated in cis. In contrast, the testis-specific FerT isomer does not oligomerize and is able to autophosphorylate in cis at two of the same three residues autophosphorylated in Fer. These results suggest that although oligomerization potentiates autophosphorylation in trans, this is apparently not necessary for Fer activation.     

Fer and Fps/Fes participate in a Lyn-dependent pathway from FcepsilonRI to platelet-endothelial cell adhesion molecule 1 to limit mast cell activation

Mast cells express the high affinity IgE receptor FcepsilonRI, which upon aggregation by multivalent antigens elicits signals that cause rapid changes within the mast cell and in the surrounding tissue. We previously showed that FcepsilonRI aggregation caused a rapid increase in phosphorylation of both Fer and Fps/Fes kinases in bone marrow-derived mast cells. In this study, we report that FcepsilonRI aggregation leads to increased Fer/Fps kinase activities and that Fer phosphorylation downstream of FcepsilonRI is independent of Syk, Fyn, and Gab2 but requires Lyn. Activated Fer/Fps readily phosphorylate the C terminus of platelet-endothelial cell adhesion molecule 1 (Pecam-1) on immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a non-ITIM residue (Tyr(700)) in vitro and in transfected cells. Mast cells devoid of Fer/Fps kinase activities display a reduction in FcepsilonRI aggregation-induced tyrosine phosphorylation of Pecam-1, with no defects in recruitment of Shp1/Shp2 phosphatases observed. Lyn-deficient mast cells display a dramatic reduction in Pecam-1 phosphorylation at Tyr(685) and a complete loss of Shp2 recruitment, suggesting a role as an initiator kinase for Pecam-1. Consistent with previous studies of Pecam-1-deficient mast cells, we observe an exaggerated degranulation response in mast cells lacking Fer/Fps kinases at low antigen dosages. Thus, Lyn and Fer/Fps kinases cooperate to phosphorylate Pecam-1 and activate Shp1/Shp2 phosphatases that function in part to limit mast cell activation.     

Closing in on the biological functions of Fps/Fes and Fer

Fps/Fes and Fer are the only known members of a distinct subfamily of the non-receptor protein-tyrosine kinase family. Recent studies indicate that these kinases have roles in regulating cytoskeletal rearrangements and inside out signalling that accompany receptor ligand, cell matrix and cell cell interactions. Genetic analysis using transgenic mouse models also implicates these kinases in the regulation of inflammation and innate immunity.     

fps/fes knockout mice display a lactation defect and the fps/fes tyrosine kinase is a component of E-cadherin-based adherens junctions in breast epithelial cells during lactation

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase implicated in vesicular trafficking and cytokine and growth factor signaling in hematopoietic, neuronal, vascular endothelial and epithelial lineages. Genetic evidence has suggested a tumor suppressor role for Fps/Fes in breast and colon. Here we used fps/fes knockout mice to investigate potential roles for this kinase in development and function of the mammary gland. Fps/Fes expression was induced during pregnancy and lactation, and its kinase activity was dramatically enhanced. Milk protein and fat composition from nursing fps/fes-null mothers was normal; however, pups reared by them gained weight more slowly than pups reared by wild-type mothers. Fps/Fes displayed a predominantly dispersed punctate intracellular distribution which was consistent with vesicles within the luminal epithelial cells of lactating breast, while a small fraction co-localized with β-catenin and E-cadherin on their basolateral surfaces. Fps/Fes was found to be a component of the E-cadherin adherens junction (AJ) complex; however, the phosphotyrosine status of β-catenin and core AJ components in fps/fes-null breast tissue was unaltered, and epithelial cell AJs and gland morphology were intact. We conclude that Fps/Fes is not essential for the maintenance of epithelial cell AJs in the lactating breast but may instead play important roles in vesicular trafficking and milk secretion.     

The protein-tyrosine kinase fer associates with signaling complexes containing insulin receptor substrate-1 and phosphatidylinositol 3-kinase

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.     

Targeted Disruption of the Murine fps/fes Proto-Oncogene Reveals that Fps/Fes Kinase Activity Is Dispensable for Hematopoiesis

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase that is functionally implicated in the survival and terminal differentiation of myeloid progenitors and in signaling from several members of the cytokine receptor superfamily. To gain further insight into the physiological function of fps/fes, we targeted the mouse locus with a kinase-inactivating missense mutation. Mutant Fps/Fes protein was expressed at normal levels in these mice, but it lacked detectable kinase activity. Homozygous mutant animals were viable and fertile, and they showed no obvious defects. Flow cytometry analysis of bone marrow showed no statistically significant differences in the levels of myeloid, erythroid, or B-cell precursors. Subtle abnormalities observed in mutant mice included slightly elevated total leukocyte counts and splenomegaly. In bone marrow hematopoietic progenitor cell colony-forming assays, mutant mice gave slightly elevated numbers and variable sizes of CFU-granulocyte macrophage in response to interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tyrosine phosphorylation of Stat3 and Stat5A in bone marrow-derived macrophages was dramatically reduced in response to GM-CSF but not to IL-3 or IL-6. This suggests a distinct nonredundant role for Fps/Fes in signaling from the GM-CSF receptor that does not extend to the closely related IL-3 receptor. Lipopolysaccharide-induced Erk1/2 activation was also reduced in mutant macrophages. These subtle molecular phenotypes suggest a possible nonredundant role for Fps/Fes in myelopoiesis and immune responses.     

Tyrosine protein kinases and spermatogenesis: truncation matters

Protein phosphorylation on serine/threonine or tyrosine residues represents a significant regulatory mechanism in signal transduction during spermatogenesis, oogenesis, and fertilization. There are several families of tyrosine protein kinases operating during spermatogenesis: the Src family of tyrosine protein kinases; the Fujinami poultry sarcoma/feline sarcoma (Fps/Fes) and Fes-related protein (Fer) subfamily of non-receptor proteins; and c-kit, the transmembrane tyrosine kinase receptor that belongs to the family of the PDGF receptor. A remarkable characteristic is the coexistence of full-length and truncated tyrosine kinases in testis. Most of the truncated forms are present during spermiogenesis. Examples include the truncated forms of Src tyrosine kinase hematopoietic cell kinase (Hck), FerT, and tr-kit. A feature of FerT and tr-kit is the kinase domain that ensures the functional properties of the truncated protein. FerT, a regulator of actin assembly/disassembly mediated by cortactin phosphorylation, is present in the acroplaxome, a cytoskeletal plate containing an F-actin network and linking the acrosome to the spermatid nuclear envelope. This finding suggests that Fer kinase represents one of the tyrosine protein kinases that may contribute to spermatid head shaping. The c-kit ligand, stem cell factor (SCF), which induces c-kit dimerization and autophosphorylation, exists as both membrane-associated and soluble. Although tyrosine protein kinases are prominent in spermatogenesis, a remarkable observation is the paucity of phenotypic alterations in spermatogenic cells in male mice targeted with Fer kinase-inactivating mutation. It is possible that the redundant functions of the tyrosine protein kinase pool present during spermatogenesis may explain the limited phenotypes of single mutant mice. The production of compound and viable mutant mice, lacking the expression of two or more tyrosine kinases, may shed light on this intriguing issue.     

High‐resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase

Fes and Fes‐related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N‐terminal Fer/CIP4 homology‐Bin/Amphiphysin/Rvs (F‐BAR) domain, a central Src homology 2 (SH2) domain, and a C‐terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine‐containing ligands to the SH2 domain. Here, the X‐ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D‐E‐pY‐E‐N‐V‐D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer‐phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I β‐turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.     

Neuropilin 1 Directly Interacts with Fer Kinase to Mediate Semaphorin 3A-induced Death of Cortical Neurons

Neuropilins (NRPs) are receptors for the major chemorepulsive axonal guidance cue semaphorins (Sema). The interaction of Sema3A/NRP1 during development leads to the collapse of growth cones. Here we show that Sema3A also induces death of cultured cortical neurons through NRP1. A specific NRP1 inhibitory peptide ameliorated Sema3A-evoked cortical axonal retraction and neuronal death. Moreover, Sema3A was also involved in cerebral ischemia-induced neuronal death. Expression levels of Sema3A and NRP1, but not NRP2, were significantly increased early during brain reperfusion following transient focal cerebral ischemia. NRP1 inhibitory peptide delivered to the ischemic brain was potently neuroprotective and prevented the loss of motor functions in mice. The integrity of the injected NRP1 inhibitory peptide into the brain remained unchanged, and the intact peptide permeated the ischemic hemisphere of the brain as determined using MALDI-MS-based imaging. Mechanistically, NRP1-mediated axonal collapse and neuronal death is through direct and selective interaction with the cytoplasmic tyrosine kinase Fer. Fer RNA interference effectively attenuated Sema3A-induced neurite retraction and neuronal death in cortical neurons. More importantly, down-regulation of Fer expression using Fer-specific RNA interference attenuated cerebral ischemia-induced brain damage. Together, these studies revealed a previously unknown function of NRP1 in signaling Sema3A-evoked neuronal death through Fer in cortical neurons.     

Role for Fes/Fps tyrosine kinase in microtubule nucleation through is Fes/CIP4 homology domain

We have previously demonstrated that Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signaling. Here we report a role for Fes tyrosine kinase in microtubule dynamics. A fibrous formation of Fes was observed in a kinase-dependent manner, which associated with microtubules and functionally correlated with microtubule bundling. Microtubule regeneration assays revealed that Fes aggregates colocalized with gamma-tubulin at microtubule nucleation sites in a Fes/CIP4 homology (FCH) domain-dependent manner and that expression of FCH domain-deleted Fes mutants blocked normal centrosome formation. In support of these observations, mouse embryonic fibroblasts derived from Fes-deficient mice displayed an aberrant structure of nucleation and centrosome with unbundling and disoriented filaments of microtubules. Our findings suggest that Fes plays a critical role in microtubule dynamics including microtubule nucleation and bundling through its FCH domain.     

Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the Fc?RI Pathway in Mast Cells

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcɛRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcɛRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcɛRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcɛRI signaling and potential regulation the actin reorganization in mast cells.Mast cells reside in connective and mucosal tissues and play a key protective role in the immune response to helminth infection (13, 21), sepsis (39), and snake or bee venoms (42). Mast cells express FcɛRI, which becomes sensitized to antigens or allergens upon immunoglobulin E (IgE) binding. Aggregation of FcɛRI by multivalent antigens causes the release of preformed mediators by degranulation and the de novo production of lipid mediators and cytokines (1, 52). Release of these mediators causes increased vascular permeability, leukocyte recruitment and activation, and inflammation (41). Aberrant mast cell activation is implicated in IgE-mediated type I hypersensitivity reactions including anaphylaxis, allergic rhinitis, and asthma (20). FcɛRI is a tetrameric receptor composed of an IgE-binding α chain and of β and γ chains containing immunoreceptor tyrosine-based activation motifs that become phosphorylated following multivalent antigen-mediated clustering of FcɛRI and activation of Src family protein tyrosine kinases (PTKs), primarily involving Lyn (51). Lyn phosphorylates and activates both positive effectors of FcɛRI signaling (e.g., Syk PTK) and key negative regulators (e.g., Shp-1 and SHIP) that serve to limit mast cell activation (28, 46, 69).Fes (the mammalian orthologue of the v-Fps and v-Fes oncoproteins from avian [57, 58] and feline [15, 56] retroviruses) and Fer are closely related PTKs that become activated following FcɛRI aggregation in mast cells (10). Surprisingly, FcɛRI-induced tyrosine phosphorylation of Fes and Fer does not require their kinase activities (55) and is almost entirely dependent on Lyn (67). Through the use of transgenic mouse models, evidence for both unique and redundant functions for Fes and Fer has been described in regulating hematopoiesis (55) and limiting the innate immune response (22, 40, 50, 72). In mast cells, we have shown that Fer promotes activation of p38 mitogen-activated protein kinase and chemotaxis of mast cells (10). We also found that Fer and Fes PTKs contribute to FcɛRI-evoked phosphorylation of platelet-endothelial cell adhesion molecule 1 (PECAM-1) (67).Each of the Fes and Fer PTKs is composed of an N-terminal regulatory domain containing a Fer-CIP4 homology (FCH) domain followed by several predicted coiled-coils (CC), a central SH2 domain, and C-terminal PTK domain (19). It is worth noting that early studies pointed toward an important role for the N-terminal domain of v-Fps for its transforming activity and membrane localization (5, 63). Several recent studies have defined the FCH and first CC domain (amino acids 1 to 300) in Fer, CIP4, and other pombe Cdc15 homology (PCH) family adaptor proteins as an F-BAR domain (also termed extended FCH or EFC domain) (reviewed in references 3 and 9). The F-BAR domain was found to constitute a novel phosphoinositide-binding domain that can promote tubulation of liposomes in vitro and membranes in vivo (27, 33, 66). The crystal structures of F-BAR domains from several PCH adaptors were recently solved (27, 59). The F-BAR module was shown to consist of a triple helical bundle that forms a homodimer, with a concave surface rich in basic residues that have recently been shown to contact phospholipids in curved membranes (16). In vitro studies using the Fer F-BAR domain have shown that the F-BAR domain binds strongly to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]; however, the Fer F-BAR is relatively weak compared with the adaptor protein FBP17 at inducing membrane tubulation (66). Liposome sedimentation assays have identified several conserved basic residues required for F-BAR domain binding to PI(4,5)P₂(66). The substitution of R113/K114 to glutamines (RK/QQ) in FBP17 reduced phosphoinositide binding by 80% (66). A recent electron cryoelectron microscopy study provided insights into binding of F-BAR dimers to flat and curved membranes via different binding faces (16). This study also confirmed that R113/K114 residues (in CIP4) constitute a site of direct interaction with the liposomes. Interestingly, microdomains of the plasma membrane rich in PI(4,5)P₂ are sites of dynamic actin assembly (47) and endocytosis (4, 31). Previous studies have described Fes localization to a variety of subcellular structures, including endocytic vesicles (71), the trans-Golgi apparatus (71), microtubules (37), and focal adhesions (44). The rapid activation of Fes and Fer PTKs upon FcɛRI aggregation on mast cells (10) would suggest that there is a mechanism by which Fes localizes at or near the plasma membrane. Phosphoinositide-binding via the F-BAR domain of Fes and Fer PTKs may promote their recruitment to the plasma membrane prior to their activation by cell surface receptors such as FcɛRI. The potential colocalization with endocytosis and actin assembly regulators may allow for regulation of receptor endocytosis or chemotaxis of mast cells by Fes/Fer PTKs. A recent study implicates Rab5 GTPase and its exchange factor RabGEF1/Rabex-5 in promoting internalization of FcɛRI following clustering by antigens (34). It is worth noting that defects in internalization of Toll-like receptor 4 and transferrin receptor were observed in Fes-deficient macrophages (48), and there is a potential role for Fes in regulating internalization of mast cell receptors.In this study, we provide novel insights into the phospholipid binding and liposome tubulating properties of the Fes F-BAR domain. Mutation of two conserved basic residues within the Fes F-BAR domain (RK/QQ) reduced phospholipid binding in vitro, and membrane localization in vivo. In transfected RBL-2H3 mast cells, the Fes harboring the RK/QQ mutation (Fes^RK/QQ) displayed reduced FcɛRI-evoked tyrosine phosphorylation compared to wild-type Fes (Fes^WT), which correlated with reduced localization to Lyn-containing membranes in mast cells. The SH2 domain of Fes was found to interact with several phosphoproteins in mast cells, including FcɛRI and HS1, an actin regulator and cortactin homologue. We found that Fes contributes to HS1 phosphorylation at C-terminal residues implicated in actin branch stabilization, and we present a model for how F-BAR-containing adaptor proteins and PTKs may coordinate actin-driven endocytosis in mast cells.     

The Fes/Fer non-receptor tyrosine kinase cooperates with Src42A to regulate dorsal closure in Drosophila

Fes/Fer non-receptor tyrosine kinases regulate cell adhesion and cytoskeletal reorganisation through the modification of adherens junctions. Unregulated Fes/Fer kinase activity has been shown to lead to tumours in vivo. Here, we show that Drosophila Fer localises to adherens junctions in the dorsal epidermis and regulates a major morphological event, dorsal closure. Mutations in Src42A cause defects in dorsal closure similar to those seen in dfer mutant embryos. Furthermore, Src42A mutations enhance the dfer mutant phenotype, suggesting that Src42A and DFer act in the same cellular process. We show that DFer is required for the formation of the actin cable in leading edge cells and for normal rates of dorsal closure. We have isolated a gain-of-function mutation in dfer (dfergof) that expresses an N-terminally fused form of the protein, similar to oncogenic forms of vertebrate Fer. dfergof blocks dorsal closure and causes axon misrouting. We find that in dfer loss-of-function mutants beta-catenin is hypophosphorylated, whereas in dfergof beta-catenin is hyperphosphorylated. Phosphorylated beta-catenin is removed from adherens junctions and degraded, thus implicating DFer in the regulation of adherens junctions.     

Semaphorin 3A contributes to distal pulmonary epithelial cell differentiation and lung morphogenesis

Rationale

Semaphorin 3A (Sema3A) is a neural guidance cue that also mediates cell migration, proliferation and apoptosis, and inhibits branching morphogenesis. Because we have shown that genetic deletion of neuropilin-1, which encodes an obligatory Sema3A co-receptor, influences airspace remodeling in the smoke-exposed adult lung, we sought to determine whether genetic deletion of Sema3A altered distal lung structure.

Methods

To determine whether loss of Sema3A signaling influenced distal lung morphology, we compared pulmonary histology, distal epithelial cell morphology and maturation, and the balance between lung cell proliferation and death, in lungs from mice with a targeted genetic deletion of Sema3A (Sema3A^-/-) and wild-type (Sema3A^+/+) littermate controls.

Results

Genetic deletion of Sema3A resulted in significant perinatal lethality. At E17.5, lungs from Sema3A^-/- mice had thickened septae and reduced airspace size. Distal lung epithelial cells had increased intracellular glycogen pools and small multivesicular and lamellar bodies with atypical ultrastructure, as well as reduced expression of type I alveolar epithelial cell markers. Alveolarization was markedly attenuated in lungs from the rare Sema3A^-/- mice that survived the immediate perinatal period. Furthermore, Sema3A deletion was linked with enhanced postnatal alveolar septal cell death.

Conclusions

These data suggest that Sema3A modulates distal pulmonary epithelial cell development and alveolar septation. Defining how Sema3A influences structural plasticity of the developing lung is a critical first step for determining if this pathway can be exploited to develop innovative strategies for repair after acute or chronic lung injury.